System lupus erythematosus (SLE) is an autoimmune disorder of complex etiology characterized by diverse immune cell dysfunction. Monogenic SLE models are uniquely useful for molecular dissection of disease pathogenesis and for testing potential therapies. TREX1/DNase III, an ER-associated exonuclease, is a negative regulator of innate immunity, and TREX1 represents one of the highest monogenic links to SLE. Curiously, most TREX1 mutations associated with SLE do not affect its DNase activity, but rather, disrupt its ER localization domain. The molecular cause for TREX1 SLE is unclear. We recently discovered a DNase- independent function of TREX1 that is required for regulating a novel ER-associated reaction. We also identified a previously unknown self-ligand derived from the ER in cells carrying TREX1 SLE mutations, as well as several small molecule inhibitors that target key enzymes that contribute to the immunopathogenesis of TREX1 SLE. Based on published and preliminary data, we hypothesize that TREX1 mutations with defective ER localization domain contribute to SLE by stimulating an ER-associated reaction that generates immunogenic self-ligands. Moreover, our biochemical data indicate that these self-ligands are free glycans. Therapeutic inhibition of this reaction or downstream innate immune signaling is predicted to ameliore TREX1 disease. We will elucidate this novel function of TREX1 with the following three specific aims. Aim 1) Genetic and biochemical dissection of a novel ER-associated reaction activated by TREX1 SLE mutations, that causes release of free glycans. We will examine TREX1 SLE and other mutations using rescue assays established in preliminary studies to identify residues in TREX1 critically required for immune activation and free glycan release. We will also biochemically dissect the interaction between TREX1 and the ER-associated glycan catabolic reaction it regulates. Aim 2) Determine how free glycan generated by TREX1 SLE mutants trigger autoimmunity. We have established an activity-based immune assay, which we will use to define structural details and immunogenic potential of free glycan as a self-ligand for TREX1 SLE and the corresponding immune signaling pathway. Aim 3) Therapeutically diminish free glycan release or inhibit TBK1 signaling in Trex1-/- mice and TREX1 SLE mutant patient cells. We will further test our hypothesis in vivo by therapeutically targeting key enzymes implicated in TREX1 SLE pathogenesis. The significance of this proposal lies with the fact that it presents an important but previously unrecognized function of TREX1 as well as identification of free glycan as a novel self-ligand in SLE. This application will also reveal and test novel targets for the treatment of SLE through 're-purposing' existing compounds developed against key enzymes involved in TREX1 SLE.